Process for preparing novenamine

ABSTRACT

Novel process for enzymatic cleavage of 3-acylamino-4-hydroxy coumarins such as are found in some antibiotic substances. Process yields amine compounds having antibiotic properties per se and/or utility as intermediates to make antibiotically active substances. Examples of compounds which can be used as starting materials in the subject process and their amine cleavage products are as follows: Novobiocin -&gt; novenamine Dihydronovobiocin -&gt; novenamine Isonovobiocin -&gt; isonovenamine Descarbamoyl novobiocin -&gt; descarbamoyl novenamine

United States Patent 91.

Sebeket a1.

[451 May 1, 1973 PROCESS FOR PREPARING NOVENAMINE [75] Inventors:Oldr'ich K. Sebek; Herman Hocksema, both of Kalamazoo, Mich.

[73] Assignee: The Upjohn Company, Kalamazoo,

Mich.

[22] Filed: Dec. 13, 1971 [21] App1.No.: 207,580

Related US. Application Data [63] Continuation-impart of Ser. No.829,757, June 2,

1969, Pat. NO. 3,652,536.

[52] 11.8. C1.. ..195/29, 195/66, 195/96 [51] Int. Cl. ..Cl2d 13/02 [58]Field of Search 195/29, 96, 51 R; 260/210 R, 210 N [56] References CitedOTHER PUBLICATIONS A. M. Rutenburg et al. New England Journal ofMedicine, Vol. 259 No. 10 pp. 469-473 Primary Examiner-Alvin E.Tanenholtz Assistant Examiner-Thomas S. Wiseman Artorney.RomanSaliwanchik et a1.

[5 7] ABSTRACT Novobiocin novenamine Dihydronovobiocin novenaminelsonovobiocin isonovenamine A descarbamoyl Descarbamoyl novobiocinnovenamine Descarbamoyl dihydronovobiocinl descarbamoyl novenamine 18Claims, No Drawings PROCESS FOR PREPARING NOVENAMINE CROSS REFERENCE TORELATED APPLICATION This application is a continuation-in-part ofpending application Ser. No. 829,757, filed on June 2, l969 now U.S.Pat. No. 3,652,536.

BRIEF SUMMARY OF THE INVENTION This invention relates to a novel processfor enzymatically cleaving 3-acylamino-4-hydroxy coumarins such as arefound in some antibiotic substances, for example, in the antibioticnovobiocin. The enzymatic cleavage of the amide bond in the antibioticnovobiocin yields 3-amino-4-hydroxy-7-0-[3-0-carbamoyl-4-O-methyl-S-deoxy-S,S-dimethyl-B-Ldyxopyranosyl]methyl coumarin, which hasbeen given the trivial name novenamine. Novenamine also can be obtainedby the enzymatic cleavage of dihydronovobiocin. Further, the subjectenzymatic process can be used to make isonovenamine from isonovobiocin;descarbamoyl novenamine from descarbamoyl novobiocin or descarbamoyldihydronovobiocin. 1

Novobiocin is an antibiotic obtainable as an elaboration product of anovobiocin-producing streptomycete. Methods for the production,recovery, and purification of novobiocin are described in U.S. Pat. No.3,049,534.

Dihydronovobiocin is an antibiotic prepared by hydrogenating novobiocinaccording to the procedures disclosed in U.S. Pat. No. 3,175,944.

Descarbamoyl novobiocin and descarbamoyl dihydronovobiocin can beprepared according to the novenamine. Thus, the applicants process forenzymatic cleavage of novobiocin to yield novenamine is the first knownprocess for accomplishing such a result and is the first known enzymaticprocess for cleaving 3- Y acylamino-4-hydroxy coumarins such as arefound in novobiocin.

DETAILED DESCRIPTION Novenamine (U-33,9l8) is prepared by enzymaticcleavage of novobiocin or dihydronovobiocin with microorganisms whichhave been identified as Arthrobacter sp., N-l8, and bacterium N-4l. Itwas discovered that these bacteria elaborate an enzyme which cleavesnovobiocin into novenamine and 4- hydroxy-3-(3-methyl-2-butenyl)-benzoicacid. This enzymatic hydrolytic cleavage is accomplished under anaerobicconditions and gives quantitative yields of the cleavage products.

A. THE MICROORGANISMS 1. Description The bacteria used according to thisinvention for the enzymatic cleavage of novobiocin have been identifiedas an Arthrobacter sp., for purposes of this application referred to asN-l8, and as bacterium N4 1 hereinafter referred to as N-4l. N-l8 andN-4l are new bacteria. One of their strain characteristics is theability to elaborate an enzyme which cleaves novobiocin. Subcultures ofthe living organisms were deposited and can be obtained from thepermanent collection of the Northern Utilization and Research Div.,Agricultural Research, U.S. Dept. of Agriculture, Peoria, 111., U.S.A.Their accession numbers in this repository are NRRL 8-3652 and NRRL13-5340, respectively.

The bacterium N-l8 is a non-motile, pleomorphic, gram-negative bacteriumof variable length, sometimes of coccoid appearance. It grows well in avariety of laboratory media containing various carbon and nitrogensources, for example, peptone, casein digests, lactalbumin digests,trypton e, trypticase, and the like. With the exception of methionineand valine, N-l 8 utilizes all the common amino acids as carbon andnitrogen source for growth, for example, aspartate, glu' tamate, lysine,arginine, histidine, proline, hydroxyproline, asparagine, glutamine,glycine, alanine, leucine, isoleucine, serine, threonine, tyrosine,phenylalanine, and tryptophan. It also utilizes novobiocin in the samemanner. The following compounds also can serve as carbon source forgrowth: ribose, xylose, glucose, fructose, galactose, gluconate,2-ketogluconate, p-hydroxybenzoate, citrate, and glycerol, but notarabinose, sorbose, maltose, sucrose, lactose, dextrin, starch,rhamnose, raffinose, inositol, sorbitol, mannitol, or p-aminobenzoate.Ammonium salts serve as adequatenitrogen sources for growth. Thebacterium N l8 grows well aerobically at 20, 28, 32, and 37C.,

but not at 42 or 5C. It is catalase-positive, but the cytochromeoxidase, indole, Voges-Proskauer and urease tests are negative. It doesnot reduce nitrate to nitrite, does not liquefy gelatin, and does notproduce H 8. Tests for arginine, lysine, and ornithine decarboxylasesare negative. The bacterium forms acids from sugars without gasevolution. It is on the basis of the above data that this bacterium hasbeen designated as belonging to the genus Arthrobacter.

The bacterium N-4l differs from N-l8 mainly in that it can use only alimited number of non-nitrogenous compounds for growth. ,It does notgrow on glucose, fructose, galactose, sorbose, arabinose, ribose,xylose, disaccharides, sorbitol, mannitol and glycols. When supplementedwith NI-l,+, it utilizes I gluconate, glycerol, lactate, C -dicarboxylicacids of the TCA cycle and a-ketoglutarate, but not citrate, acetate,for mate, malonate, salicylate, m-Ihydroxybenzoate, or phenylacetate. Itutilizes certain L-amino acids of the aspartic family (aspartate,homoserine, threonine, isoleucine, lysine, asparagine), glutamate family(glutamate, proline, glutamine), pyruvate family (alanine, leucine), andB-methylaspartate, and phenylalanine. It is a non-motile gram-negativerod with uniform terminal swelling as seen by electron microscope. Itreduces nitrate to nitrite, is catalase-positive but indole, cytochromeoxidase, urease, hydrogen sulfide and Voges-Proskauer negative, andproduces no pigment. Gelatin is not liquefied, milk not peptonized andmethylene blue is not reduced. Satisfactory growth of the microorganismis obtained at 24-37C.

The cytosine-guanine (CG) content of its isolated DNA is percent whichcorresponds to the values for the taxonomically ill-definedhigh-percent-CG Arthrobacter-Alcaligenes group (see Antonie vanLeeuwenhoek 36:193-207, 1970). On the basis of these properties(especially its inability to utilize sugars and its CG content), itstaxonomic position fits best the genus Alcaligenes. A direct comparisonto the authentic type strain A. methalcaligenes which it resembles mostclosely (non-motile), was also made but the two organisms differedconsiderably in their metabolic patterns (A. methalcaligenes does notcleave novobiocin.

2. Maintenance The bacteria are maintained on slants of agar mediumcontaining 0.5 percent tryptone, 1 Gold Label Brer Rabbit molasses, 1percent glycerol, 0.25 percent yeast extract, and 1.5 percent agar in 1liter distilled water stored at C. and transferred in 2-4 weekintervals. The bacteria also can be maintained on media of comparativelysimple composition, for example, 0.2 percent Difco Casitone agar aloneor supplemented with 0.1 percent glucose and 0.05% NlLCl and inorganicsalts, or 0.1 percent glucose and 0.05 percent yeast extract. Complexmedia, for example, brain heart infusion broth, do not contribute to theelaboration of the novobiocin-cleaving enzyme which is designated hereinas novobiocin acylase. Preferably, the bacteria are stored over liquidnitrogen at about -196C.

3. Growth Conditions The cells of bacteria N-l8 and N-41 can be grownand produce novobiocin acylase in a variety of media consisting ofvarious carbon and nitrogen sources as long as good growth of thebacterium is obtained. Suitable carbon and nitrogen sources are yeastextract, NZ-Amine B (Sheffields enzymatic digest of casein), Casitone(Difco), glycerol, NH Cl, and the like. The media can be supplemented,advantageously, with inorganic salts, for example, Kl-l PO MgSO NaCl,FeCl FeSO and the like. A preferred medium consists of 0.8% NZ-Amine B,0.5% KH PO 0.05% NaCl, 0.02% MgSO; 7H O and 0.001% FeSO (pl-l 7.6).

The hydrolytic (enzymatic cleavage) property of the bacteria isevidenced by use of a test depending on the fact that novenamine israpidly converted to a brownreddish pigment when exposed to the air. Ifsuch a color develops upon incubation of a particular bacterial isolatewith novobiocin, or another suitable substrate, as herein disclosed, theisolate has the desirable hydrolyzing property because novenamine ispresent. A reliable bacterial selection can be carried out in thefollowing way. The culture is grown in the above liquid NZ-Amine Bmedium in a shaken flask overnight at room temperature. An aliquot isthen serially diluted in test tubes and a loopful of each dilution isstreaked on NZ-Amine B agar (composition as above) and incubated untilcolonies develop. A number (usually lO to 50) of discrete andwell-defined colonies are selected and each transferred with steriletoothpicks on (1) NZ- Amine B agar (of the above composition), and (2) amedium containing inorganic salts (as above) and 0.1 0.2 percentnovobiocin (Na salt) as the only C and N source, and incubated for 2-3days at room temperature. After that time, colonies grown on novobiocinwhich will form brown-reddish halos, are those which cleave novobiocinto novenamine (and which in turn was converted to the pigment) and,therefore, contains the novobiocin hydrolyzing enzyme. These coloniesare then picked from the NZ-Amine B agar (instead of from novobiocinagar to minimize the inhibitory effect of the novobiocin products on theviability of the isolate), and grown again in NZ-Amine B liquidovernight, aliquots distributed in 1 ml. portions in plastic vials andstored under nitrogen. Modifications of this procedure are possible.

1. NZ-Amine B broth is seeded, incubated overnight, an aliquot of theheavily grown culture is serially diluted and the more highly dilutedsamples (to favor the outgrowth of separate colonies) are streaked onNZ-amine B agar supplemented with 0.1 percent novobiocin. After 2-3 daysof incubation, those colonies which have the characteristicbrown-reddish halos are then transferred directly as desired.

2. An isolate is grown in NZ-Amine B broth overnight, a drop of thegrown culture is placed on the surface of an agar plate containing 0.1percent novobiocin (with or without additional salts) and incubatedovernight at room temperature. If the brown-reddish color develops inthe area of application, novobiocin cleavage (hydrolysis) has takenplace.

3. The time for this color development may be shortened to 20-120minutes if more enzyme is supplied for the reaction. In this instance,cells from 10-50 ml. of the culture which has been grown overnight asunder (2), are harvested by centrifugation and washed once with water. Asmall volume (0.2 0.5 ml.) is mixed with the same volume of 0.2 percentnovobiocin. The suspension is exposed to air (air may be replaced byoxygen) and either allowed to stand or to shake at room temperatureuntil the characteristic color is noted. The time needed for this colordevelopment depends not only on a sufficient amount of oxygen but alsoon the amount of enzyme supplied (the heavier the cell suspension is,the faster the pigmentation appears) but reliable reading can be madewithin a 20-120 minute incubation time.

B. THE ENZYMATIC NOVOBIOCIN The enzymatic cleavage of novobiocinproceeds as follows:

CLEAVAGE OF CH CH3 1 (Moiety C and B) Novrnamino By substituting in theabove process descarbamoyl novobiocin, or isonovobiocin, for novobiocin,there is obtained descarbamoyl novenamine, and isonovenamine,respectively.

The cells of N-l8 or N-4l are grown for l 3 days at an incubationtemperature of about 25-37C. The preferred growth time is about 1 day atan incubation temperature of about 28C. Advantageously, the cells areagitated and aerated in the medium during growth.

The bacterial cells are harvested from the growth medium,advantageously, by centrifugation. The harvested cells are washed withwater and resuspended, advantageously, in a minimum amount of water.This material then can be suspended in an aqueous solution ofnovobiocin, for example, sodium novobiocin, where the cleavage processtakes place. As disclosed above, the cleavage process is conducted underanaerobic conditions. Such anaerobic conditions can be achieved in thelaboratory by various means known in the art, for example, overlayingthe reaction mixture with an airimpervious substance, for example,toluene, or, flushing the reaction mixture with nitrogen or other inertgas. Anaerobic conditions in processing equipment larger than laboratorysize can be achieved by various methods known in the art.

Generally, the cleavage of novobiocin by the enzyme formed by bacteriumN-l8 or N41 is complete in about 18-42 hours at room temperature. Highertemperatures, for example, 34C. to about 37C., will shorten the reactiontime; whereas lower temperatures, for example C. to about 24C., willlengthen the reaction time. The cleavage is preferably conducted at roomtemperature. Higher concentrations of the cells (containing higherconcentrations of the desired enzyme) also shorten the reaction time.

Upon completion of the cleavage process, the bacterial cells areseparated from the reaction mixture. These cells of N-l8 and N-4l arequite stable and not readily inactivated during the cleavage process.Thus,

. the same cells can be used 4 6 times over in cleavage processes asdescribed above. The cells can also be lyophilized and stored forprolonged periods of time at low temperatures (preferably belowfreezing) but some loss of the novobiocin-cleaving ability occurs underthese conditions. The cells are separated from the reaction mixture bycentrifugation or filtration. Centrifugation is preferred. The amount ofnovenamine in the cell-free supernatant can be ascertained by thefollowing method: An aliquot (0.1 0.5 ml.) of supernatant is diluted,adjusted to pH 2.0 with 3 N HCl, extracted with butyl acetate, theextract diluted with 2 volumes of acidified (3 N HCl) ethanol (pH 2.0),and the amount of novenamine quantitatively determined by its absorptionat 295 mp. The amount of novenamine is calculated according to thefollowing formula:

(CD 5 my. X Dilution/35.4) mg. novenamine/ml.

*35.4 equals 0D,, 5 mp of l mg. novenamine/ml. (=a) OD Optical DensityNovobiocin concentrations of from about 1 2.5 percent are cleaved tonovenamine (and Moiety A) in yields of 91-100 percent in about 1 day bythe abovedescribed cleavage process.

Hydrolysis of novobiocin to novenamine (and 3-isopententyl-4-hydroxybenzoic acid) also has been found to be broughtabout by cell-free enzyme containing extracts of N-l8 and/or N-4l. Theseextracts are dition (50 mg./ml.) of deoxyribonuclease l (WorthingtonBiochemical Corp., Freehold, N.J.), the debris and unbroken cells arecentrifuged off, and the supernatant is dialyzed against 0.05 Mphosphate buffer (pH 7.5) overnight at 6C. Other methods which can beused to provide the hydrolytic enzyme in cell-free form included thelysis of the cells by lysozyme (l mg./ml., Sigma Chemical Co., St.Louis, Mo.), and sonication (Raytheon magnetostrictive oscillator, 10kc) for 5 15 minutes at 4-l0C. As disclosed above, the hydrolysis ofnovobiocin has to be performed in the absence of oxygen since novenaminesolution, if exposed to air, gradually darkens, a reaction typical ofaromatic amines.

Dihydronovobiocin also can be substituted for novobiocin in theabove-disclosed enzymatic cleavage processes to yield novenamine;descarbamoyl novobiocin can be substituted for novobiocin to yielddihydronovobiocin are extractable at acid pH. For example, novenamine isrelatively insoluble in chlorinated lower-alkanes, e.g., methylenechloride, chloroform, ethylene dichloride, and the like; waterimmiscible esters, e.g. ethyl acetate, butyl acetate, amyl acetate, andthe like; and water-immiscible higher alcohols. Novenamine is unstablein the presence of ox-.

ygen in solution under alkaline conditions.

Novenamine can be recovered from the enzymatic cleavage reaction by avariety of procedures, for example, the use of adsorbents, andcrystallization from so]- vents. The first step of any such process, advantageously, involves the removal of the bacterial cells;advantageously, this is done by centrifugation or filtrating of anacidified suspension. In a preferred process, the cleavage supernatantor filtrate containing novenamine, as well as various impurities such assome of the uncleaved starting material, i.e., novobiocin ordihydronovobiocin, is adjusted to an acidic pH of about 2 to 6, thenextracted with a solvent in which novenamine is not soluble. Any of thesolvents disclosed above can be used; butyl acetate is the preferredsolvent. The extraction of the acidified cleavage supernatant removesunreacted substrates, for example, novobiocin or dihydronovobiocin. aswell as the cleaved hydroxybenzoic acid, thus leaving a supernatantwhich can be freeze-dried to a relatively pure preparation of novenamineacid addition salt. This preparation of novenamine can be used inenvironments where higher purity preparations of novenamine are notessential. Also, this relatively pure preparation of novenamine can beacylated to form antibacterially-active novenamine acylates, ashereinafter described.

Higher purity novenamine preparations can be obtained by subjecting arelatively pure preparation of novenamine, as disclosed above, tocrystallization procedures from a solvent such as methanol. For example,the water in a relatively pure preparation of novenamine hydrochloridecan be removed by azeotropic distillation, for example, using absoluteethanol, and novenamine then crystallized from ab solute ethanol andether to afford a purified preparation of novenamine.

Suitable acids for adjusting the pH of the cleavage supernatant, asdisclosed above, are strong mineral or organic acids for example,hydrochloric, hydrobromic, phosphoric, sulfuric, formic, oxalic, and thelike. Freeze-dried novenamine will be isolated as the salt of the acidused. For example, if hydrochloric acid is used to adjust the pH of thecleavage supernatant, then novenamine will be isolated as thehydrochloride salt. Novenamine is a very weak base and the salts are notvery stable. This salt can be converted to the free base form ofnovenamine by suspending it in tetrahydrofuran and adding about oneequivalent of triethylamine or by dissolving it in slightly moistacetone, or by prolonged drying under reduced pressure or bycrystallization from methanol. Other salts of novenamine then can bemade from the free base. Salts of novenamine can be used for the samebiological purposes as the free base.

Novenamine also can be recovered from the cleavage supernatant,disclosed above, by converting the novenamine to an N-acylate. Anexample of such a procedure is as follows: (1) acidify cleavagesupernatant to about pH 2.5 with a mineral acid, for example,hydrochloric acid, (2) extract with a solvent for novobiocin, forexample, butyl acetate, (3) adjust pH to about 8.0 with a base, forexample, 6 N sodium hydroxide, and (4) add a halide or anhydride of aselected carboxylic acid to form novenamine N- acylate. A nitrogenatmosphere should be maintained in the above-disclosed process once thereaction mixture is adjusted to an alkaline pH.

Mineral acids other than hydrochloric, which were disclosed previously,can be used in the above process. Also, other solvents for novobiocin,disclosed previously, can be used for butyl acetate in the aboveprocess. Bases which can be used in the above process in place of sodiumhydroxide are sodium bicarbonate or sodium carbonate, lithium orpotassium hydroxide, bicarbonates, or other bases or basic salts ofmetals. In addition water soluble tertiary amines such as trialkylamines (triethylamine) or pyridine, quinoline and the like may be used.

Novenamine can also be recovered from aqueous solutions by adsorption ona surface active absorbent, for example, deoxygenated decolorizingcarbon or decolorizing resins, and eluting the adsorbed material with asolution of water and acetone.

lsonovenamine and descarbamoyl novenamine can be recovered fromenzymatic cleavage process solutions in the same manner as disclosedabove or by obvious modifications thereof.

Novenamine, descarbamoyl novenamine, and isonovenamine can be acylatedby the procedures disclosed in application Ser. No. 829,757 to yieldcompounds having antimicrobial activity.

The following examples are illustrative of the process and products ofthe present invention, but are not to be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1 Enzymatic Cleavage of Novobiocin A loopful of the cells from aslant of bacterium N-l 8, NRRL B-3652, or N-4l, NRRL B-5340, isinoculated into 250 ml. Erlenmeyer flasks containing 100 ml. of a mediumwhich consists of the following ingredients: 0.8% NZ-Amine B, 0.05% KHPO 0.05% NaCl, 0.02% MgSO -7H O and 0.01% FeSO per. liter deionizedwater, pH 6.8 7.8. The flasks are incubated at 28C. on rotary shaker.After 18 hours of incubation, when the bacterial population has almostreached its maximum density, the bacterial cells are harvested bycentrifugation, washed once with distilled water and resuspended inwater to give a ml. final volume. Oneml. fractions are then pipettedinto a series of 5 test tubes, novobiocin as the sodium salt is added,and the final volume in each test tube is adjusted to 3 ml. The tubesare flushed with nitrogen, closed tightly and incubated on areciprocating shaker. After 20 hours of incubation at room temperature,the cells are removed by centrifugation and the cell-free supernatantsare analyzed for the presence of novenamine by the procedure disclosedpreviously to give the following results:

Novobiocin Novenamine (initial concentration) Formed mgJml. YieldsExample 2 Isolation of Novenamine Hydrochloride A 4.5 liter portion ofcell-free liquid, from a larger scale enzymatic cleavage process, asdescribed in Example 1, containing cleavage product from 12 grams ofnovobiocin, is acidified with 15 ml. of 6 N hydrochloric acid andextracted once with 1 liter of methylene chloride, and once with 800 ml.of methylene chloride. The solvent extract is discarded and the aqueousmaterial is freeze-dried; yield, 10 g. of crude novenamine powder whichgives an instantaneous purple color with ninhydrin in pyridine.

An 800-mg. aliquot of the above preparation of novenamine isrecrystallized from absolute ethanol, hydrogen chloride, and ether afterremoval of water by distillation from absolute ethanol; yield, 400mg. ofnovenamine hydrochloride crystals having a melting point 300C. dec.

A purified preparation of novenamine has the following elementalanalysis:

Analysis:

Calc'd for C,,l-l N,O,Cl:

C, 49.49; H. 5.47; N, 6.08; CI, 7.69.

Found: C, 49.26; H, 6.61, N, 5.81.

Novenamine hydrochloride has the following strong IR bands in mineraloil mull: (in cm") 3420 (sh shoulder) I645 3350 1606 3190 1575 2920(oil) 1540 2860 (oil) 1485 2650 1455 1707 M35 (oil) 1375(oil) 1130 (sh)l340 l l l5 1265 1083 1255 1000 1210 CH: CH3

-0 Q 0 -O 0113K 1 \O l CH3 a 1-1 OH OH Isonovenamine is characterized bythe following structural formula:

H CONH2 Example 4 By substituting a substantially cell-free extract ofbacteria N-l 8 and/or N-4l for the living bacteria in Examples 1 and 3,there are obtained the enzymaticcleavage products characterized inExamples 1, 2, and

We claim:

1. A process for preparing novenamine, the compound having thestructural formula:

dihydronovobiocin with an amide cleaving enzyme elaborated by abacterium selected from the group consisting of Arthrobacter sp. N-l8NRRL B-3652,'and

bacterium N-4l NRRL 3-5340.

2. A process for preparing novenamine, according to claim 1, whereinnovobiocin is incubated with bacterium Arthrobacter sp. N-l8 NRRLB-3652, under anaerobic conditions.

3. A process for preparing novenamine, according to claim 1, whereinnovobiocin is incubated with bacterium N-4l NRRL 3-5340, under anaerobicconditions.

4. A process for preparing isonovenamine, the compound having thestructural formula:

OH oooNHz which comprises incubating isonovobiocin with an amidecleaving enzyme elaborated by a bacterium selected from the groupconsisting of Arthrobacter sp. N-l8 NRRL b3652, and bacterium N-41 NRRLB- 5. A process for preparing isonovenamine, according to claim 4,wherein isonovobiocin is incubated with bacterium Arthrobacter sp. N-18NRRL B-3652, under anaerobic conditions.

6. A process for preparing isonoyenamine, according to claim 4, whereinisonovobiocin is incubated with bacterium N-4l NRRL B-5340, underanaerobic conditions.

7. A process for preparing descarbamoyl novenamine, the compound havingthe structural formula:

which comprises incubating descarbamoyl novobiocin or descarbamoyldihydronovobi-ocin with an amide cleaving enzyme elaborated by abacterium selected from the group consisting of Arthrobacter sp. N-l8NRRL 8-365 2, and bacterium N-41 NRRL B-S 340.

8. A process for preparing novenamine, according to claim 1, whereinnovobiocin or dihydronovobiocin is incubated with a substantiallycell-free extract of a bacterium selected from the group consisting ofArthrobacter sp. N-l8 NRRL 13-3652, and bacterium N-4l NRRL 8-5 340.

9. A process for preparing novenamine, according to claim 8, whichcomprises incubating novobiocin or dihydronovobiocin with asubstantially cell-free extract from the bacterium Arthrobacter sp. N-l8NRRL B- 3652.

10. A process for preparing novenamine, according to claim 8, whichcomprises incubating novobiocin or dihydronovobiocin with asubstantially cell-free extract from the bacterium N-41 NRRL 13-5340.

cum

(His

11. A process for preparing isonovenamine, according to claim 4, whereinisonovobiocin is incubated with a substantially cell-free extract of abacterium selected from the group consisting of Arthrobacter sp. N-l8NRRL 8-3652, and bacterium N-4l NRRL 13-5340.

12. A process for preparing isonovenamine, according to claim 11, whichcomprises incubating isonovobiocin with a substantially cell-freeextract from the bacterium Arthrobacter sp. N-l8 NRRL B- 13. A processfor preparing isonovenamine, according to claim 11, which comprisesincubating novenamine, according to claim 14, which comprises incubatingdescarbamoyl novobiocin or dihydronovobiocin with a substantiallycell-free extract of the bacterium Arthrobacter sp. N-l 8 NRRL 8-3 652.

16. A process for preparing descarbamoy novenamine, according to claim14, which comprises contacting descarbamoyl novobiocin ordihydronovobiocin with a substantially cell-free extract of thebacterium N-4l NRRL 8-5340.

17. A process for preparing descarbamoyl novenamine, according to claim7, wherein descarbamoyl novobiocin or descarbamoyl dihydronovobiocin isincubated with bacterium Arthrobacter sp. N-l 8 NRRL 8-3652, underanaerobic conditions.

18. A process for preparing descarbamoyl novenamine, according to claim7, wherein descarbamoyl novobiocin or descarbamoyl dihydronovobiocin isincubated with bacterium N-4l NRRL 8-5340, under anaerobic conditions.

2. A process for preparing novenamine, according to claim 1, whereinnovobiocin is incubated with bacterium Arthrobacter sp. N-18 NRRLB-3652, under anaerobic conditions.
 3. A process for preparingnovenamine, according to claim 1, wherein novobiocin is incubated withbacterium N-41 NRRL B-5340, under anaerobic conditions.
 4. A process forpreparing isonovenamine, the compound having the structural formula:which comprises incubating isonovobiocin with an amide cleaving enzymeelaborated by a bacterium selected from the group consisting ofArthrobacter sp. N-18 NRRL b3652, and bacterium N-41 NRRL B-5340.
 5. Aprocess for preparing isonovenamine, according to claim 4, whereinisonovobiocin is incubated with bacterium Arthrobacter sp. N-18 NRRLB-3652, under anaerobic conditions.
 6. A process for preparingisonovenamine, according to claim 4, wherein isonovobiocin is incubatedwith bacterium N-41 NRRL B-5340, under anaerobic conditions.
 7. Aprocess for preparing descarbamoyl novenamine, the compound having thestructural formula: which comprises incubating descarbamoyl novobiocinor descarbamoyl dihydronovobiocin with an amide cleaving enzymeelaborated by a bacterium selected from the group consisting ofArthrobacter sp. N-18 NRRL B-3652, and bacterium N-41 NRRL B-5340.
 8. Aprocess for preparing novenamine, according to claim 1, whereinnovobiocin or dihydronovobiocin is incubated with a substantiallycell-free extract of a bacterium selected from the group consisting ofArthrobacter sp. N-18 NRRL B-3652, and bacterium N-41 NRRL B-5340.
 9. Aprocess for preparing novenamine, according to claim 8, which comprisesincubating novobiocin or dihydronovobiocin with a substantiallycell-free extract from the bacterium Arthrobacter sp. N-18 NRRL B-3652.10. A process for preparing novenamine, according to claim 8, whichcomprises incubating novobiocin or dihydronovobiocin with asubstantially cell-free extract from the bacterium N-41 NRRL B-5340. 11.A process for preparing isonovenamine, according to claim 4, whereinisonovobiocin is incubated with a substantIally cell-free extract of abacterium selected from the group consisting of Arthrobacter sp. N-18NRRL B-3652, and bacterium N-41 NRRL B-5340.
 12. A process for preparingisonovenamine, according to claim 11, which comprises incubatingisonovobiocin with a substantially cell-free extract from the bacteriumArthrobacter sp. N-18 NRRL B-3652.
 13. A process for preparingisonovenamine, according to claim 11, which comprises incubatingisonovobiocin with a substantially cell-free extract from the bacteriumN-41 NRRL B-5340.
 14. A process for preparing descarbamoyl novenamine,according to claim 7, wherein descarbamoyl novobiocin or descarbamoyldihydronovobiocin is incubated with a substantially cell-free extract ofa bacterium selected from the group consisting of Arthrobacter sp. N-18NRRL B-3652, and bacterium N-41 NRRL B-5340.
 15. A process for preparingdescarbamoyl novenamine, according to claim 14, which comprisesincubating descarbamoyl novobiocin or dihydronovobiocin with asubstantially cell-free extract of the bacterium Arthrobacter sp. N-18NRRL B-3652.
 16. A process for preparing descarbamoy novenamine,according to claim 14, which comprises contacting descarbamoylnovobiocin or dihydronovobiocin with a substantially cell-free extractof the bacterium N-41 NRRL B-5340.
 17. A process for preparingdescarbamoyl novenamine, according to claim 7, wherein descarbamoylnovobiocin or descarbamoyl dihydronovobiocin is incubated with bacteriumArthrobacter sp. N-18 NRRL B-3652, under anaerobic conditions.
 18. Aprocess for preparing descarbamoyl novenamine, according to claim 7,wherein descarbamoyl novobiocin or descarbamoyl dihydronovobiocin isincubated with bacterium N-41 NRRL B-5340, under anaerobic conditions.